The present invention is concerned with new methods and reagents for coupling proteins and a wide variety of non-protein affinity ligands by strong covalent chemical bonds to solid-phase supports for the preparation of solid-phase catalysts and biospecific adsorbents.
Biospecific adsorbents have become very important tools for the isolation of biologic macromolecules, while immobilized enzymes have found wide use as durable, solid-phase catalysts. Such materials are prepared by chemically linking enzymes, inhibitors, or other biospecific compounds to a solid-phase support. Various methods and reagents have been described for the purpose of coupling, but each has its peculiar drawbacks, including: (a) weak chemical bonding; (b) ionic bonds; (c) necessity for strong conditions such as high temperature and pH to drive the reaction; (d) ride reactions; and (e) limited range of chemical groups with which the coupling reagent can react.
Previously coupling reagents include the reagent most widely utilized as a coupler, cyanogen bromide, as described by Jacoby et al. in Methods Enzymol. 34, 1974. This reagent is reacted with support such as agarose and cellulose in strongly alkaline solution where numerous side reactions occur. The cyanogen bromide-activated support subsequently reacts poorly with nucleophiles other than alkylamines, a severe limitation on the kinds of enzymes, ligands and other compounds which can be coupled. The coupling bonds, probably isourea bonds, are only moderately stable, and substantial amounts of the bound substance may "bleed" under practical conditions. This instability limits the useful life of the adsorbent or solid-phase catalyst and may add undesirable or potentially toxic contaminants to the product. Moreover, these isourea bonds are positively charged at neutral pH and can cause non-specific adsorption.
In contrast, reagents of a type that can provide strong covalent bonds between diverse supports and various nucleophilic groups of ligands under mild conditions have been employed in the dye industry since the introduction of "reactive" dyes. The most versatile and widely employed of these coupling reagents is trichloro-s-triazine (TsT). This reagent contains three reactive chlorines, the first of which is displaced to give a substituted dichloro-s-triazine (DsT), and the second to give a disubstituted monochloro-s-triazine (MsT). Conditions for controlled substitutions by various nucleophiles have received comparatively little study.
Like cyanogen bromide, TsT has been reacted with supports in strongly alkaline aqueous media where hydrolytic side reactions predominate. In the procedure as described by Kay et al. in Nature 217:641, (1968), the amount of TsT which reacts with support and that which hydrolyzes are competitive functions of temperature, pH and reagent concentration, and the extent of activation of the support cannot be predicted accurately. Moreover, the activated support is also subject to hydrolysis. Recently, Kay and Lilly have introduced the less reactive coupler, 2-amino-4,6-dichloro-s-triazine, as discussed in Biochim. Biophys. Acta. 198:276 (1970). However, in alkaline aqueous media, this reagent is subject to the same drawbacks as TsT during the activation reaction and, in addition, vigorous conditions are required for coupling the ligands. The major problems with such prior art methods are: (1) couplers are used empirically as others have used cyanogen bromide; (2) although triazine coupling provides strong bonds between support and proteins, no attention has been previously paid to competitive hydrolytic side reactions and to the introduction of adsorptive ionic sites; (3) the amount of ligand incorporated cannot be accurately controlled or predicted; and (4) the procedures are not satisfactory for the incorporation of small ligands in organic phase.
In order to circumvent the problem of TsT hydrolysis and to perform step-wise reactions at individual chlorines of TsT, by the present invention there have been developed methods for reaction in non-aqueous media. The critical conditions were found to include appropriate polar organic solvents and suitable organic bases to neutralize the HCl generated. With polyol supports like cellulose or cross-linked agarose, as described by Porath et al. in J. Chromatogr. 60:167 (1971), reactions with TsT have been found to occur smoothly and predictably in organic phase to give a (DsT)-dichloro-s-triazine substituted support. This activated support could be reacted in organic phase at one or both remaining chlorines, depending upon the nucleophiles involved and the reaction conditions.
It has also been found, in accordance with the present invention, that weak nucleophiles such as aniline react at room temperature in organic solvent with one of the chlorines of the DsT-support, leaving a single site for subsequent reaction. In fact, the initial "activation" reaction could be well controlled without side reactions to give a DsT-support; and a subsequent reaction with a weak nucleophile could be performed without side reactions to give an MsT-support. Finally, the single remaining chlorine of the MsT-support is less reactive than the DsT-support or TsT and is less susceptible to hydrolysis at moderate pH and temperature. Therefore, it can be reacted with nucleophiles in organic or in aqueous solution under mild conditions of pH and temperature to give the desired products.
In J. Biol. Chem. 252, 3578 (1977) Abuchowski et al. have reported the covalent attachment of polyethylene glycol (PEG) to proteins using TsT as a linking agent. While the coupling of an aliphatic alcohol to TsT in organic phase is the initial reaction in both the Abuchowski et al. procedure and in the present invention, this initial coupling is the entire extent of any similarity.
In their initial coupling reaction, Abuchowski et al. use benzene as a solvent for both PEG and TsT and use Na.sub.2 CO.sub.3 to neutralize the HCl generated. This method, while adequate only for the coupling of TsT and PEG in solution phase, is not satisfactory for the coupling of TsT and any of the solid-phase supports utilized in affinity preparations, such as Sepharose, cellulose or polyvinyl alcohol. Benzene has proved to be a poor medium for reactions involving Sepharose and other hydrophilic polymers, most likely because of the non-polar nature of benzene. A major problem in the case of Sepharose or other polymers initially in aqueous phase is the transfer to benezene. It has also been observed that benzene seems to have an adverse effect on Sepharose structure. Furthermore, benzene is not a particularly good solvent for TsT, as discussed in a "s-Triazines and Derivatives," in The Chemistry of Heterocyclic Compounds (E. M. Smolin and O. Rapoport, eds.). Interscience Publishers, Inc., New York, Vol. 13, 1959.
Neutralization of the HCl generated is inefficient when the base is insoluble, as is the case of Na.sub.2 CO.sub.3 in benzene. Inefficient neutralization of the HCl could permit conversion of the alcohol to the corresponding alkyl chloride, a side reaction known to occur under these conditions. Also, incorporation of chlorine into the Sepharose matrix is likely to impart undesirable properties.
By the present invention, there has been achieved the ability to circumvent many of the problems inherent in the Abuchowski et al. procedure, by conducting the initial activation reaction in dioxane or other organic solvents and by neutralizing the HCl generated with a soluble organic base such as N,N-diisopropylethylamine or other tertiary amine which does not form an insoluble complex with TsT in organic phase. Of the several reaction media which have been tried, dioxane was found to be best as it is both compatible with Sepharose and other hydrophilic polymers and is also a superior solvent for TsT. The use of a soluble organic base is also novel, and selection of the correct base is critical as most form insoluble complexes with TsT.
After the initial activation step, the two procedures are obviously different. Abuchowski et al. are satisifed with empirical coupling of dichloro-s-triazine-PEG to proteins in aqueous phase under alkaline conditions. The reference also states that a considerable excess of activated PEG must be used because hydrolysis of the second chlorine of the triazine occurs readily. This is a common feature of triazine reactions in aqueous or mixed aqueous-organic phase and it prevents accurate control and predictability of the reaction.
Unreacted and hydrolyzed PEG-dichloro-s-triazine are, of course, soluble and and separable from the PEG-coupled protein. This is not the case, however, if one wants a broader objective, i.e., coupling of an insoluble solid-phase compound such as cellulose-dichloro-s-triazine to a ligand where any side reactions such as hydrolysis affect the desired product. In the procedure of the present invention, control is established by replacing the second triazine chloride by a weak nucleophile such as aniline. This leaves a single chlorine available for reaction with nucleophilic groups of the proteins or other substances to be immobilized. This single chlorine can be replaced quantitatively by aliphatic amines under mold conditions, yet it does not hydrolyze readily below pH 9 at room temperature.
Various supports (polyols) can be reacted with TsT in accordance with the present invention, but all such supports must be rid of water prior to the initial reaction. Where suitable, drying can be performed by heating in vacuo, or water may be displaced from the support by washing with dry, miscible organic solvents such as 1,4-dioxane, acetonitrile and similar solvents. In addition, the parameters of the reaction conditions can be altered to effect changes in the amount of TsT which reacts with the support, including changes in reaction temperatures, reagent concentration, and the duration of the reaction.
The reaction of TsT with the support in organic phase requires the presence of a suitable organic base. The bases generally employed in analogous acyl chloride type reactions such as triethylamine, pyridine, N-ethyl morpholine, and lutidine, were found to form insoluble complexes with TsT in organic solvents. However, it was found that tertiary amines such as N,N-dimethylaniline and N,N-diisopropylethylamine did not form such complexes and performed satisfactorily.